Francisella tularensis is a highly infectious Category A bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Due to the ease of aerosol dissemination of this organism and the minimal inoculum (s10 bacteria) necessary to cause severe disease, F. tularensis has been weaponized for use in biowarfare. Critical to Francisella's pathogenesis are its ability to replicate within macrophages, the primary niche for replication in vivo, and to subvert the host immune response. Many genes contribute to the ability of bacterial pathogens to replicate within the host, but distinct immunomodulatory virulence factors, which are not required for replication, often play crucial roles in evading host immune responses. Unfortunately, relatively little is known about which genes F. tularensis uses to subvert host defenses and how these genes are regulated. We recently employed a powerful global in vivo negative selection screen in mice to identify genes required for the pathogenesis of Francisella. This approach resulted in the identification of 164 genes that are required for virulence, 44 of which appear to encode novel virulence factors. Among the genes encoding novel virulence factors were 2 that we showed are dispensable for bacterial replication within macrophages but play critical roles in suppressing the macrophage cell death response, an important host defense. We will1 screen mutants for each of the other 162 genes identified in our screen to identify those that are dispensable for bacterial replication in macrophages, but alter macrophage defense responses. We will determine how they contribute to the pathogenesis of virulent F. tularensis at the molecular level. Elucidation of the molecular mechanisms of action of critical F. tularensis virulence factors will significantly enhance our understanding of Francisella pathogenesis as well as common themes in host-pathogen interactions. This work will generate data that will lay the groundwork for the next generation of therapeutics and vaccines against potential biowarfare agents and emerging infections.